WO1998046725A2 - Vaccins vivants attenues diriges contre des ichtyopathogenes - Google Patents

Vaccins vivants attenues diriges contre des ichtyopathogenes Download PDF

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WO1998046725A2
WO1998046725A2 PCT/US1998/007066 US9807066W WO9846725A2 WO 1998046725 A2 WO1998046725 A2 WO 1998046725A2 US 9807066 W US9807066 W US 9807066W WO 9846725 A2 WO9846725 A2 WO 9846725A2
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bacterium
recited
fish
vaccine
step comprises
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PCT/US1998/007066
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WO1998046725A3 (fr
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Ronald L. Thune
Richard K. Cooper
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Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College
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Priority to EP98918062A priority Critical patent/EP0973865A2/fr
Priority to IL13232798A priority patent/IL132327A0/xx
Priority to AU71055/98A priority patent/AU7105598A/en
Priority to US09/402,695 priority patent/US6350454B1/en
Publication of WO1998046725A2 publication Critical patent/WO1998046725A2/fr
Publication of WO1998046725A3 publication Critical patent/WO1998046725A3/fr
Priority to IL132327A priority patent/IL132327A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00—Drug, bio-affecting and body treating compositions
    • Y10S424/827—Bacterial vaccine for fish

Definitions

  • This invention pertains to fish vaccines, particularly to certain live-attenuated bacterial vaccines against fish pathogens.
  • BACKGROUND ART Immune responses to live vaccines are generally of greater magnitude and of longer duration than those produced by killed or subunit vaccines.
  • a single dose of a live-attenuated vaccine can provide better protection against later infection by the wild-type organism, because the attenuated organism persists and metabolizes within the host, and in some cases may replicate in the host for a time. See, e.g. , M. Roberts et al. , "Salmonella as Carriers of Heterologous Antigens," pp. 27-58 in O'Hagan (ed.), Novel Delivery Systems for Oral
  • Vaccines (1994). Live vaccines better elicit cell-mediated immune responses, which can have a crucial role in controlling infections by intracellular pathogens. Injectable vaccines are impractical in most commercial fish culture due to extensive pond or cage production techniques, large numbers of individual animals, and low value per individual animal. Prior immersion or oral delivery of killed vaccines to fish has yielded inconsistent results. The invasion, persistence, and replication of live-attenuated vaccines has the potential to provide effective, inexpensive vaccines. R. Thune et al. , "Studies on Vaccination of Channel Catfish, Ictalurus punctatus, against Edwardsiella ictaluri" pp. 11-23 in D. Tave et al.
  • auxotrophic bacterium is a nutritional mutant requiring one or more growth factors to survive and replicate. Certain nutrients have limited availability in vertebrate tissues. A bacterium from an otherwise pathogenic species will be attenuated if it is made auxotrophic for such a limited nutrient. These auxotrophic mutants are potentially useful as live-attenuated vaccines.
  • adenine auxotrophic Salmonella pur A mutants are less effective as vaccines than aroA mutants, possibly because pur A mutants are overly attenuated due to the extremely low availability of adenine in mammalian tissues.
  • the channel catfish (Ictalurus punctatus) is the most important aquaculture species in the United States.
  • R. Thune "Bacterial Diseases of Catfish," Chapter 57 (pp. 511-520) in Stoskopf, M.K. (ed.), Fish Medicine (1993) reviews the major bacterial diseases encountered in commercial catfish aquaculture, the most serious of which is enteric septicemia of catfish (ESC).
  • Edwardsiella ictaluri the bacterium that causes ESC, was first described in 1979 after isolation from catfish farms in Georgia and Alabama. Since then it has been reported in every state that produces channel catfish commercially.
  • Edwardsiella ictaluri was isolated from 46.2% of the channel catfish cases submitted to aquatic animal diagnostic laboratories in Alabama, Louisiana, and Mississippi during 1987-89.
  • the various Edwardsiella ictaluri strains that have been examined to date have been serologically and biochemically homogenous.
  • killed bacterins have been evaluated as vaccines against ESC.
  • a protective response has been inconsistent in field trials using killed preparations, and it has been suggested that prior, sub-clinical exposure of vaccinated fish to E. ictaluri during periods in which temperatures were not conducive to disease may have been an important factor in establishing this response; and that a similar response might not be seen in naive fish without a similar sub-clinical exposure. Thune et al. (1994).
  • a strong cell-mediated immune response could provide a more effective vaccination against ⁇ SC ⁇ both for the above reasons, and because E. ictaluri is a facultative intracellular pathogen.
  • Injection of a killed preparation with an adjuvant is one way to stimulate cell-mediated immunity (CMI), but because of the large numbers, small size, and low economic value of individual fish, this route of vaccination is not practical in commercial catfish production.
  • CMI cell-mediated immunity
  • Live-attenuated strains of pathogenic bacteria could potentially generate a strong CMI.
  • attenuated strains of invasive pathogens may be delivered via oral and immersion routes, making their administration more economical.
  • no previous vaccines have been reported to stimulate cell-mediated immunity against E. ictaluri.
  • hybrid striped bass (Morone saxatilis x Morone chrysops) is a rapidly expanding aquaculture industry in the United States, the Mediterranean region, and southeast Asia, including Taiwan.
  • hybrid striped bass production increased from 3750 tons in 1994 to 7000 tons in 1996 (Hybrid Striped Bass Growers Association, personal communication). This fish is adapted for culture in both fresh and brackish water, resulting in the development of significant production of this hybrid species in coastal areas worldwide.
  • coastal hybrid striped bass farms are located in Louisiana, Texas, and Florida.
  • Pasteurella piscicida has seriously restricted the expansion of commercial aquaculture in warm water coastal areas.
  • Pasteurella piscicida has recently been renamed Photobacterium damsela subspecies piscicida. The historical nomenclature Pasteurella piscicida is used here.
  • Pasteurellosis was relatively unknown outside of Japan prior to 1990. In Japan pasteurellosis has caused losses in excess of $20 million annually in cultured yellowtail.
  • the recent growth of coastal aquaculture in the United States and in the Mediterranean region has created ideal conditions for this highly pathogenic, halophilic organism.
  • 32 cases of heavy mortality in coastal hybrid striped bass farms have been reported in the last five years (Louisiana Aquatic Animal Diagnostic Lab case records), with two farms closing as a result of P. piscicida losses.
  • Pasteurellosis is an acute, rapidly developing disease. Antibiotic treatments have often been impractical or ineffective. In addition, P. piscicida has quickly developed resistance to certain antibiotics. An effective vaccine would circumvent these problems. However, previous vaccinations of hybrid striped bass against P. piscicida using killed autogenous bacterins, Alpharma (Bellevue. WA) and AquaHealth (Ontario, Canada), delivered by immersion or injection, have not provided satisfactory results in the field (Dr. R. Ariav, personal communication).
  • Known host fishes of Pasteurellosis include the following: the temperate basses (Family Percichthyidae), including the white bass Morone americanus, the striped bass Morone saxatilis, and their hybrids; the sea basses (Family Serranidae), including the Japanese sea bass Lateolabrax japonicus, the Asian sea bass Lates calcarifer, and the European sea bass, Dicentrarchus labrax; the jacks (Family Carangidae), including yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex; the filefishes (Family Beautystidae), including the oval filefish Navodan modestus; and the seabream (Family Sparidae), including the black seabream Acanthopagrus shlegeli, the red sea bream Pagrus major, and the gilthead seabream Sparus aurata.
  • the temperate basses Feamily Percichthy
  • ALB reduced mortality to challenge from 81.3 % in controls to 25.3 % in vaccinated fish.
  • the FKB and HKB reduced mortality to 57.3% and 78.7% , respectively.
  • ALB increased phagocytic activity over controls from 4.0% to 19.0% , compared to 4.8% with HKB and 8.0% with FKB, while the increase in antibody level was similar for all three treatments.
  • the ALB vaccines of this study were produced by serial passage on growth media, and are thus potentially susceptible to spontaneous reversion to virulence. The overall genetic change needed for reversion in such cases can be quite low — even a single point mutation may suffice.
  • United States patent no. 5,536,658 discloses a chondroitinase-attenuated Edwardsiella strain used as vaccine for catfish and other fish susceptible to Edwardsiella infection, administered by immersion, injection, or in feed.
  • United States patent no. 5,498,414 discloses attenuated Aeromonas salmonicida strains used as immersion vaccines for chinook salmon and rainbow trout.
  • the attenuated strains were reported to lack a functional A-protein, a component of the cell membrane.
  • the A- protein gene could be disrupted, for example, by insertion of a gene encoding an antigenic protein of another fish pathogen, thus potentially allowing the attenuated Aeromonas salmonicida to vaccinate fish against two pathogens.
  • E. Dunn et al. "Vaccines in Aquaculture: The Search for an Efficient Delivery
  • P. Homchampa et al. "Construction and Vaccine Potential of an aroA mutant of Pasteurella haemolytica," Veterinary Microbiology, vol. 42, pp. 35-44 (1994) discloses the use of an attenuated Pasteurella haemolytica mutant with an aroA mutation to immunize mice, as a model for a cattle vaccine against bovine pneumonic pasteurellosis.
  • L. Vaughan "An Aromatic-Dependent Mutant of the Fish Pathogen Aeromonas salmonicida Is Attenuated in Fish and Is Effective as a Live Vaccine against the Salmonid Disease Furunculosis," Infection and Immunity, vol. 61, pp. 2172-2181 (1993) discloses that an attenuated Aeromonas salmonicida with an aroA mutation was not virulent when injected intramuscularly into Atlantic salmon; and that intraperitoneal vaccination with the attenuated strain conferred protective immunity to brown trout against infection by a virulent A. salmonicida strain. See also L.
  • J. Plumb et al. "Vaccination of Channel Catfish, Ictalurus punctatus (Rafinesque), by Immersion and Oral Booster against Edwardsiella ictaluri," J. Fish Diseases, vol. 16, pp. 65- 71 (1993) discloses a formalin-killed Edwardsiella ictaluri immersion vaccine that produced humoral immunity in Ictalurus punctatus, with or without a subsequent oral booster.
  • these vaccines may be used not only to vaccinate fish against Edwardsiella ictaluri or Pasteurella piscicida, but also to serve as vectors to present antigens from other pathogens to the fish immune system, thereby serving as vaccines against other pathogens as well, with no risk of infection by reversion to the virulent form of the pathogen in which the antigen occurs naturally.
  • LSU- ⁇ 1 (ATCC No. 55947); derived from 93-146, except AaroA::Tn903 (Km r ) as described below.
  • LSU- ⁇ 2 (ATCC No. 55948); derived from 93-146, except ApurA: :Tn903
  • pEI22 Ap, pBluescript derivative with 2789 base pair segment of the E. ictaluri chromosome inserted in the Not I site and containing the aroA gene; present work.
  • Edwardsiella ictaluri strain 93-146 The parental organism for the two live-attenuated vaccines is Edwardsiella ictaluri strain 93-146, which was isolated from catfish undergoing an ESC epizootic in north Louisiana in May 1993. Edwardsiella ictaluri strain 93-146 was confirmed using standard microbiological methods and API 20e test strips, generating a code of 4004000. The strain was sensitive to oxytetracycline, Romet, erythromycin, nitrofurantoin, and kanamycin. No genetic modifications were made in E. ictaluri strain 93-146 prior to construction of the two attenuated mutants described below.
  • This mutant strain, LSU-E1 (ATCC No. 55947), had a 259 bp deletion within the 1284 base pair aroA gene. Base pairs 541-801 of the aroA gene were deleted.
  • the mutant required supplementation with aromatic metabolites, namely ⁇ ra-aminobenzoic acid, di- hydroxybenzoic acid, and hydroxybenzoic acid, in E. ictaluri minimal media (EI-MM), as otherwise described in L. Collins et al. , "Development of a Defined Minimal Medium for the Growth of Edwardsiella ictaluri, " Applied and Environmental Microbiology, vol. 62, pp. 848- 852 (1996).
  • An inserted marker gene for this mutant was a 1697 bp BamHl kanamycin resistance fragment from Tn90J.
  • the deletion mutant was constructed using the following technique.
  • An E. ictaluri genomic library was initially created in an intermediate cloning vector, the phagemid ⁇ ZapTM (Stratagene Inc., La Jolla, CA), carrying plasmid pBKCMV.
  • E. coli E. coli.
  • the entire insert was sequenced, and was determined to be 2789 bp long.
  • the complete E. ictaluri aroA gene was determined to occur at base pairs 1238 to 2524 of the insert; the gene was 1287 nucleotides long, with 68% identity to the E. coli aroA gene.
  • the fragment was sub-cloned in pBluescript (Stratagene, Inc., La Jolla, CA) to facilitate subsequent manipulations, and the resulting plasmid was named p ⁇ I22. Sequence analysis indicated that the restriction endonuclease N ⁇ rl cut pEI22 at base pairs 1780 and 2038 of the insert in pEI22, but would not cut the pBluescript vector.
  • pEI22 was digested with the endonuclease N ⁇ rl to create a 259 bp deletion in the aroA gene, and also to linearize pEI22.
  • the N ⁇ rl overhangs of the linearized plasmid were filled in using Klenow fragment, and the kanamycin marker gene was inserted by blunt end ligation, creating pEI23.
  • the 2364 bp fragment containing the mutated gene was ligated into the T-protein based suicide vector pGP704 for transfer to wild-type E. ictaluri by RP4-mediated conjugation and homologous recombination, creating pEI24.
  • Transconjugates were selected on media containing kanamycin, with colistin added to counter-select against the E. coli donor. Because the suicide vector contained an ampicillin resistance marker, kan r transconjugates were replica- plated to media containing ampicillin to establish ampicillin sensitivity, to verify that the vector had not been maintained. After isolation of Kan7amp s colonies, the strain E. ictaluri LSU-E1 (ATCC No.
  • primer aro-5 designed to anneal to base pairs 1507-1524 of the negative strand of the aroA gene
  • primer kan+ designed to anneal to base pairs 1399-1417 of the positive strand of the kan marker sequence
  • LSU-E1 construct Stability of the LSU-E1 construct was demonstrated by growing it in 30 successive passages in culture tubes containing 5 mL Brain Heart Infusion Broth (BHI) without kanamycin. A 100 ⁇ L aliquot from overnight cultures was used to inoculate each subsequent tube. At pass 30, two 5 mL cultures were pelleted in a centrifuge, suspended in 100 mL saline, and spread on EI-MM to detect revertants to the wild-type phenotype, i.e., the ability to grow on EI-MM without aromatic metabolite supplementation. Prior to spreading on EI- MM, an aliquot was removed and serially diluted to determine colony forming units (cfu)/mL in the concentrated suspensions. A total of 5.67 x 10'° cfu's plated on minimal media after 30 passes in BHI yielded no revertant colonies.
  • BHI Brain Heart Infusion Broth
  • a sample of the bacterium LSU-E1 was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on April 9, 1997, and was assigned ATCC Accession No. 55947. This deposit was made pursuant to a contract between ATCC and the assignee of this patent application, Board of Supervisors of Louisiana State University and Agricultural and Mechanical College. The contract with ATCC provides for permanent and unrestricted availability of the bacterium to the public on the issuance of the US. patent describing and identifying the deposit or the publication or the laying open to the public of any U.S. or foreign patent application, whichever comes first, and for availability of the progeny of the bacterium to one determined by the U.S.
  • Escherichia coli was grown at 37°C with Luria-Bertani (LB) broth and agar plates following the method of Sambrook et al.. Molecular Cloning: a Laboratory Manual, Cold
  • Edwardsiella ictaluri was grown at 28°C with BHI and agar plates, or with trypticase soy agar (TSA) II plates with 5% sheep blood.
  • LambdaZapTM Express bacteriophage (Stratagene, La Jolla, CA) were grown in E. coli XLl-Blue MRF' (Stratagene) with NZYMTM agar plates and NZYMTM top agarose.
  • EI-MM broth and agar plates with and without supplemented adenine (25 ⁇ g/ml) were used for nutritional characterization of E. ictaluri strains.
  • the API 20E system (bioMerieux Vitek, Hazel wood, MO) was used for species identification and biochemical characterization of E. ictaluri strains. Conjugations between E. ictaluri and E. coli were grown at 28 °C on LB plates. The F ' episome was maintained in E. coli XLl-Blue MRF' with tetracycline selection at 12.5 ⁇ g/ml. Ampicillin at 200 ⁇ g/mL was used to maintain pBluescript (Stratagene), pGP704, and their derivatives. Kanamycin at 50 ⁇ g/mL was used to maintain plasmids derived from the pBK-CMV phagemid and plasmids carrying Tn903.
  • Colistin at 10 ⁇ g/mL was used for counterselection against E. coli SM10 ⁇ pir following conjugations.
  • E. coli XLl-Blue MRF was spread on LB plates with 100 ⁇ L of 100 mM IPTG and 40 ⁇ L of 2% X-gal.
  • Edwardsiella ictaluri genomic DNA was prepared from overnight 100 mL cultures using a modification of the protocol by Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (1994). Bacteria were pelleted by centrifugation at 5000 x g for 10 minutes and resuspended in 9.5 mL TE (10 mM Tris and 1 mM EDTA, pH 8.0). Cells were lysed in 0.5 % SDS and 100 ⁇ g/mL of proteinase K at 50°C for 2 hours.
  • the aqueous layer containing genomic DNA was transferred to a fresh tube, and an equal volume of 24: 1 chloroform/isoamyl alcohol was added. The aqueous phase was separated again by centrifugation and transferred to a fresh tube. DNA was precipitated by addition of sodium acetate (pH 5.5) to 0.3 M followed by addition of an equal volume of isopropanol. The precipitated DNA was spooled from the aqueous-isopropanol interface using a sterile glass rod, washed in 70% ethanol, and resuspended in 3 mL of TE buffer.
  • Both E. coli and E. ictaluri were prepared for electroporation using the protocol of Ausubel et al. (1994). Washed E. coli were transfected by electroporation in 0.2 cm cuvettes at 2.5 kV and 25 ⁇ F, with the pulse controller set at 200 ohms. Washed E. ictaluri were electroporated using the same protocol at 1.75 kV. Bacteria were recovered for 1 hour in BHI broth at 37 °C (E. coli) or 28 °C (E. ictaluri) before being spread on selective media.
  • E. ictaluri purA gene An 1104 base pair fragment of the E. ictaluri purA gene was amplified from genomic E. ictaluri DNA using PCR primers derived from conserved regions of the E. coli purA gene, as determined by alignments of published purA gene sequences. See Wolfe et al. , "Nucleotide Sequence and Analysis of the purA Gene Encoding Adenylosuccinate Synthetase of Escherichia coli K12, " J. Biol. Chem. , vol. 263, pp. 19147-19153 (1988); see also Mantsala et al.
  • PCR reactions were conducted on a Perkin Elmer DNA Thermal Cycler 480 using AmpliTaq DNA Polymerase at pH 8.5, with a magnesium concentration of 1.5 mM, 125 ng of template DNA per reaction, a 0.25 ⁇ M concentration of each primer, and a 30 ⁇ M concentration of each dNTP. Cycle conditions were 95 °C for 30 seconds, 53 °C for 45 seconds, and 72 °C for 45 seconds for 35 cycles; with an initial denaturation step at 95°C for 2 minutes, and a final extension step at 72°C for 10 minutes. To increase total PCR product yield, a second PCR was carried out using the product from the first PCR as the template (0.5 ⁇ L of a 1/10 dilution) under the same conditions.
  • the purA PCR product was purified using the Elu-QuickTM DNA Purification Kit to remove excess primers and dNTPs.
  • Primers for both PCR and sequencing were synthesized using solid-phase cyanoethyl phosphoramidate chemistry on a Perkin Elmer/ Applied Biosystems DNA Synthesizer Model 394.
  • Agarose gels were prepared for Southern hybridization using the protocol of Ausubel et al. (1994), and DNA was transferred to Nytran PlusTM 0.45 ⁇ m nylon membranes using a
  • PosiBlotTM 30-30 pressure blotter and pressure control station (Stratagene).
  • the 1104 base pair E. ictaluri purA PCR fragment was denatured by boiling, and was labeled directly with horseradish peroxidase using the ECLTM direct nucleic acid labeling and detection system (Amersham Life Science, Arlington Heights, IL). Labeled PCR fragments were used as a probe for plaque hybridization and Southern hybridization. Prehybridization, hybridization, and stringency washes were all performed in tubes at 41 °C according to the ECLTM protocol using a hybridization oven with an integral rotisserie device, followed by chemiluminescent detection.
  • the E. ictaluri genomic library was constructed by cloning E. ictaluri genomic DNA after partial digestion with Sau3A I into the BamH I site of ⁇ ZapTM Express.
  • MRF was transfected with the library according to the ⁇ ZapTM Express protocol, and was spread on three plates containing approximately 11,000 plaque forming units (pfu) per plate. Plaques were transferred to Nytran PlusTM 0.45 ⁇ m nylon membranes (Schleicher & Schuell), and phage DNA was released, denatured, and fixed to the membranes according to the manufacturer's protocol. The library was screened by DNA hybridization using the labeled
  • Plasmid pEI14 DNA was digested with Not I, and the resulting 3.5 kb fragment was ligated into Not I-digested CIP-treated pBluescript.
  • E. coli XLl-Blue MRF was transfected with the ligation mix by electroporation, and was spread on LB/Amp/Tet plates with IPTG and X-gal for blue-white screening. The resulting 6.5 kb plasmid was designated pEI15.
  • Plasmid pNK2859 DNA was digested with BamH I to isolate a 1.7 kilobase fragment containing the Tn903 kanamycin resistance gene, and pEI15 DNA was digested with Nar I to remove a 598 base pair fragment from the purA gene sequence. Both digests were treated with Klenow fragment in the presence of dNTPs to fill in the sticky ends.
  • the 5.9 kilobase band from pEI15 was blunt end-ligated to the 1.7 kilobase band from pNK2859, and E. coli XLl-Blue MRF was transfected with the ligation mix by electroporation and spread on LB/Amp/Kan plates. This 7.6 kilobase plasmid was designated pEI16.
  • Plasmid pEI16 DNA was digested with Not I to remove the 4.6 kilobase insert, and the Not I sites were filled in using Klenow fragment. This fragment was blunt end-ligated into the EcoR V site of pGP704, and E. coli CC118 ⁇ pir was transfected with the ligation mix by electroporation and spread on LB/Amp/Kan plates. The resulting 8.5 kilobase plasmid carrying the ApurA: :Km r construct and over 2200 bp of flanking E. ictaluri chromosomal sequence was designated p ⁇ I17. Plasmid pEI17 was subsequently transferred to E. coli SM10 ⁇ pir by electroporation.
  • the cell densities of an E. ictaluri culture grown to mid-log phase, and of an overnight SM10 ⁇ pir culture were estimated by measuring absorbance at 600 nm, and the two cultures were mixed in 5 mL of 10 mM MgS0 4 in a 2: 1 ratio (approximately 1.3 x 10 8 E. ictaluri and 6.5 x 10 7 E. coli).
  • Plasmid p ⁇ I17 was transferred into E ictaluri using the RP4 origin of transfer by conjugation with E coli SM10 ⁇ pir as the donor strain
  • the plasmid pEI17 did not persist in E ictaluri, as judged by sensitivity to ampicillin, plasmid preparations, and negative PCR results using internal pGP704 primers from the ampicillin resistance gene
  • 1865 kanamycin-resistant E ictaluri colonies were isolated from approximately
  • E ictaluri LSU-E2 Stability of the adenine auxotrophic phenotype in E ictaluri LSU-E2 (ATCC No 55948) was demonstrated by growing it in 30 successive passages in culture tubes containing 5 mL BHI without kanamycin The final two 5 mL cultures were pelleted by centrifugation in a swinging bucket rotor at 4750 x g for 10 minutes The pellets were resuspended in 0 9 mL of sterile 0 9% saline, and nine 100 ⁇ L ahquots from each were spread on E ictaluri minimal media plates, and were checked for growth 4 days later Serial dilutions of the suspension were dropped in 20 ⁇ L ahquots on BHI plates, and were counted 2 days later
  • Edwardsiella ictaluri 93-146 genomic DNA, and genomic DNA from LSU-E2 were used as templates for a series of PCR amplifications to confirm the genotype of LSU-E2 Plasmid pEI17 DNA was used as a positive control for the reactions Primer concentrations of 0 5 ⁇ M, dNTP concentrations of 100 ⁇ M, and 100 ng of template DNA were used for each reaction Cycle conditions were 95°C for 30 seconds, 59°C for 1 mmute, and 72°C for 3 minutes for 35 cycles, with an initial denaturation step at 95°C for 2 minutes, and a final extension step at 72°C for 10 minutes
  • Amplification of a portion of the Tn903 kanamycin resistance gene using primers 903Kan+ and 903Kan- yielded the predicted 624 base pair band from mutant chromosomal DNA, indicating that the Tn90 gene had been successfully incorporated into the LSU-E2 chromosome. Only non-specific bands were obtained from control, wild-type E. ictaluri. Amplification using primers pBRAmp+ and pBRAmp- yielded only non-specific bands from both LSU- ⁇ 2 and wild-type E. ictaluri, indicating the ampicillin resistance gene from pEI17 had not been incorporated into the chromosome.
  • Plasmid pEI17 DNA which was used as a positive control for this reaction, yielded the predicted 709 base pair band for the ampicillin resistance gene.
  • Primers PurAUl and 903Kan+ amplified the 3' end of the Tn90J insert and flanking purA gene sequence, resulting in a 1191 base pair fragment from LSU-E2 DNA and no amplification for wild-type DNA.
  • Primers PurAMl l and Tn903M2 yielded a 587 base pair fragment from LSU-E2 DNA containing the 5' end of the Tn903 insert and flanking purA gene sequence; comparable results were again negative for wild-type DNA. Sequencing of the
  • ATCC American Type Culture Collection
  • ictaluri strain caused 96.7 - 100% mortality at intraperitoneal injection doses of 10 5 cfu and 10 6 cfu, 72.4 - 100% at 10 4 cfu, 56.6 - 73.3 % at 10 3 cfu, and 43.3 - 56.6% at 10 2 cfu.
  • Mortality with the mutant strains could only be demonstrated when — 10 7 bacteria were injected per fish, a dose that, with minimal replication in the host, approximated total bacterial load in fish that die from a wild- type infection.
  • Both mutants were avirulent when administered by immersion at a dose of 10 7 cfu/ml, while immersion in 10 7 cfu/mL of the wild type caused 63.3 - 100% mortality. Both mutants were avirulent at oral doses of 10 8 cfu per fish, while wild-type bacteria caused 20.7 - 26.7% mortality at the same dose.
  • SPF channel catfish fingerlings were stocked at a rate of ten per tank, and were randomly divided into three treatment groups with three tanks per treatment.
  • One of the treatment groups was experimentally infected by immersion with wild-type E. ictaluri, one was experimentally infected with LSU-E2 E. ictaluri, and the third was experimentally infected with LSU-E1 E. ictaluri. Wild-type, LSU-E2, or LSU-E1 E.
  • ictaluri bacterial culture was added directly to the flow-through tanks at doses corresponding to approximately 3.5 x 10 7 cfu/mL for wild-type, 6.7 x 10 6 cfu/mL for LSU-E2, and 2.1 x 10 7 cfu/mL for
  • one fish was removed from each tank and was euthanized by transfer to water containing 1 g/L MS- 222.
  • the study was extended and fish were also collected on days 4 and 5 (only two fish were sampled on day 5).
  • One fish was collected from each tank prior to experimental infections for a zero hour sample.
  • samples were taken of liver, spleen, head kidney, and trunk kidney from each fish. The samples were suspended in 0.5 mL sterile 0.9% saline solution, weighed, and pulverized.
  • the resulting suspension was serially diluted in 0.9% saline solution in triplicate using 96-well plates, and 20 ⁇ L aliquots were dropped on BHI plates for quantification. Colonies were counted after incubation for 48 hours. Edwardsiella ictaluri and other bacterial species were identified using the API 20E system.
  • Adenine auxotrophic E. ictaluri strain LSU-E2, or aromatic auxotrophic E. ictaluri strain LSU-E1 was isolated from the internal organs in all of the immersion- exposed channel catfish sampled from 2 hours post-exposure to 48 hours post-exposure, indicating that the invasive capabilities of the attenuated bacteria were intact. However, the infection was limited and all tissues tested were cleared of viable auxotrophic E. ictaluri by 3 days post-exposure. At each sampling time bacterial concentrations in the tissues were significantly higher for wild-type E. ictaluri than for either LSU-E2 or LSU-E1. Maximum tissue levels for the auxotrophic E. ictaluri strain were 10 4 cfu/gm of tissue at 2 hours post- exposure. All fish tested before the experimental infections were negative for E. ictaluri in any tissues.
  • the wild-type exposure caused rapid penetration of the host. Head and trunk kidneys had the highest numbers of bacteria per gram of tissue. By 2 hours post-exposure there were approximately 10 4 cfu/gram, and by 6 hours post-exposure approximately 10 6 . Numbers increased to approximately 10 7 per gram from 3 to 5 days post-exposure. All fish cultured positive at all sampling times. Bacterial concentrations were slightly lower in the spleen than in the kidneys throughout the study, but the difference was not statistically significant. Bacterial counts in the liver did not rise as quickly, and were significantly lower than the other tissues.
  • Vaccination Dose Booster Dose (at 4 Challenge % Mortality ⁇ St. Dev. weeks if given)
  • the vaccinates had significant less mortality than the non- vaccinated fish.
  • vaccinates without a booster had similar mortality rates as vaccinates at the 4-week challenge.
  • vaccinates with a booster had significantly less mortality at the 8-week challenge.
  • the best results were obtained with an initial vaccination at 10 7 cfu/mL, followed by a booster 4 weeks later at either 10 7 cfu/mL or 10 8 cfu/mL.
  • E. ictaluri purA A second study was conducted to evaluate the efficacy of LSU-E2 as a vaccine.
  • One hundred eighty juvenile SPF channel catfish were stocked at a rate of 15 per tank, and were randomly divided into two treatment groups with six tanks per treatment.
  • One treatment group was vaccinated with LSU-E2 E. ictaluri by immersion, and the other group was not vaccinated.
  • Two hundred ml of an LSU-E2 overnight culture was added directly to vaccinated tanks, and water flow was stopped for 15 minutes following initial exposure. Bacterial concentration in the water was approximately 3.65 x 10 7 cfu/ml.
  • both vaccinated and non-vaccinated treatments were experimentally infected with wild-type E. ictaluri by immersion exposure.
  • Edwardsiella ictaluri bacterial culture was added directly to the flow-through tanks for a final bacterial concentration of approximately 5.3 x 10 7 cfu/L in the water. Water flow was stopped for 15 minutes following initial exposure and then resumed. Mortalities were recorded each 24 hour period after experimental infection until day 26 post-exposure.
  • Bacterial samples were taken from the trunk kidney of each dead fish, and were cultured on TSA II plates with 5 % sheep blood to confirm E. ictaluri as the cause of death.
  • Feeding activity remained normal following immersion vaccination of channel catfish with LSU- ⁇ 2; no mortalities followed the vaccination. All mortalities following immersion exposure to wild-type E. ictaluri were culture positive for E. ictaluri from the trunk kidney.
  • Non-vaccinated tanks had a final average mortality of 33.3 % , significantly higher (P ⁇ 0.01) than the average 11.1 % mortality for the vaccinated tanks.
  • the average mortality in the non-vaccinated tanks was significantly higher (P ⁇ 0.01) than the average for the vaccinated tanks for each day from day 7 post-exposure through the end of the study.
  • E. ictaluri strain 93-146 has not yet been evaluated for virulence in hosts other than channel catfish.
  • E. ictaluri is somewhat host specific for the North American freshwater catfish family Ictaluridae, with isolates reported from channel catfish (Ictalurus punctatus), white catfish (Ictalurus catus), and brown bullhead (Ictalurus nebulosus).
  • Experimentally exposed golden shiners Notemigonus crysoleucas
  • largemouth bass Micropterus salmoides
  • bighead carp Aristichthys nobilis
  • E. ictaluri has now been reported from several natural outbreaks in non-ictalurid tropical fish, including green-knife fish (Eigemannia virescens), danio (Danio devario), Rosy barbs (Puntius conchonius), and walking catfish (Clarius batrachus).
  • Green-knife fish Eigemannia virescens
  • danio Danio devario
  • Rosy barbs Piertius conchonius
  • walking catfish Clarius batrachus
  • European catfish Siluris glanis
  • rainbow trout Oncorhynchus mykiss
  • chinook salmon Oncorhynchus tshawytscha
  • the vaccines reported here are expected to be effective in protecting other species susceptible to infection by E. ictaluri, because the manner in which the vaccine strains were attenuated is not tailored to any specific host.
  • the insertion/deletion mutant aroA was subcloned into the EcoRI site of the suicide plasmid pGP704, and transferred to a wild-type P. piscicida strain by electroporation and homologous recombination.
  • Analogous techniques will be used to create a ApurAv.kan mutant of P. piscicida.
  • Mutant AaroAwkan (or ApurAv.kan) colonies from double cross-over homologous recombinations were selected on a defined media with aromix (aromatic amino acids and para- amino-benzoic acid) or adenine supplementation, together with selection for kanamycin resistance.
  • Putative mutants were replica plated to minimal media (with kanamycin) without nutrient supplementation to confirm the auxotrophic phenotype. Genetic conformation of the mutant was be confirmed using polymerase chain reaction to amplify fragments with primers internal to the inserted kanamycin marker and primers from the flanking aroA sequence.
  • a sample of the bacterium LSU-P2 was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on April 9, 1998, and was assigned ATCC Accession No. xxxxx. This deposit was made pursuant to a contract between ATCC and the assignee of this patent application, Board of Supervisors of Louisiana State University and Agricultural and Mechanical College. The contract with ATCC provides for permanent and unrestricted availability of the bacterium to the public on the issuance of the US. patent describing and identifying the deposit or the publication or the laying open to the public of any U.S. or foreign patent application, whichever comes first, and for availability of the progeny of the bacterium to one determined by the U.S.
  • Attenuated E. ictaluri and P. piscicida retain their invasive properties and can be administered by immersion, these attenuated strains are ideal candidates to use as vectors in delivering heterologous antigens for vaccination.
  • the genetic manipulation techniques have been established for attenuated Salmonella strains. The same general techniques will be used here. A number of different genes from bacteria, viruses, parasites, and mammals have been successfully expressed in attenuated Salmonella, and the recombinant strains have been used to immunize small animals. See review in Roberts et al. (1994).
  • the same techniques as described above will be used to create aroA deletion mutants or purA deletion mutants, where the inserted sequences contain both the kanamycin resistance gene to facilitate selection, and also a gene encoding the heterologous antigen.
  • the gene for the heterologous antigen is placed under the control of the native promoter for the aroA gene or purA gene, as appropriate, to ensure that the antigen is expressed and is "seen" by the fish immune system during the relatively brief residence of the attenuated strain in the fish before it is cleared.
  • the aroA promoter will be active in conditions where the attenuated E. ictaluri is starved for aromatic amino acids; and the purA promoter will be active in conditions where the attenuated E.
  • ictaluri is starved for adenine.
  • the gene for the heterologous antigen may be placed under the control of a constitutive promoter, such as the constitutive E. ictaluri methylase gene promoter.
  • a constitutive promoter such as the constitutive E. ictaluri methylase gene promoter. See Jie Zhang, "Identification, Cloning and Sequence of a Methylase Gene from Edwardsiella ictaluri," M.S. Thesis (Louisiana State University, Baton Rouge, 1995).
  • the E. coli alkaline phosphatase promoter is also known to be constitutive in E. ictaluri.
  • These vaccines are preferably administered to relatively young fish, most preferably to relatively young fish raised in a specific pathogen free environment, so that the fish will not have already developed immunity to the wild type of the carrier strain (e.g., wild-type E. ictaluri or wild-type P. piscicida). Such pre-existing immunity could cause the vaccine strain to be cleared from the fish too quickly to establish an immune response to the heterologous antigen.
  • the carrier strain e.g., wild-type E. ictaluri or wild-type P. piscicida
  • heterologous antigens to be used in this aspect of the invention include the several membrane associated proteins from Channel Catfish Virus (CCV). These are encoded by open reading frames 6, 7, 8, 10, 19, 46, 51, and 59 of the CCV genome as described by A. Davison, "Channel Catfish Virus: A new type of herpesvirus," Virology, vol. 186, pp. 9-14 (1992); and are all available for subcloning and characterization from a CCV genomic DNA library constructed in the cosmid pHC 79 in our laboratory, L.
  • CCV Channel Catfish Virus
  • CCV Chiannel catfish herpesvirus
  • Additional antigens that may be used in this aspect of the invention include, for example, the glycoprotein and ribonucleoprotein gene of infectious hematopoietic necrosis virus (H. Engelking et al., "Glycoprotein from Infectious Hematopoietic Virus (IHNV) induces protective immunity against five IHNV types," J. Aquatic Animal Health, vol. 1, pp 291-300 (1989); J. Koener et al. , "Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus," J. Virology, vol. 61, pp. 1342-1349 (1987); and L.
  • the aroA gene or purA gene be disrupted by deletion of a large portion of the gene, as such a mutation minimizes the risk of a spontaneous reversion to virulence.
  • the deletion should preferably encompass at least 10 bases, more preferably at least 100 bases.
  • other mutations known in the art that will inactivate or greatly reduce the activity of the gene may also be used. Examples of such mutations include point mutations within the gene or promoter (insertion, deletion, or alteration); insertion of a transposon, mini-transposon, or other sequence into the gene or promoter; or altering the promoter to one that is expressed at much smaller levels. Any such mutation that effectively makes the bacterium an auxotroph will function in the present invention, although it is highly desirable to use a mutation with a minimal risk of reversion to wild type.
  • a "protective amount" of an attenuated bacterium is an amount that, when administered to a fish as a vaccine, induces a degree of immunity sufficient to reduce to a statistically significant degree the susceptibility of the fish to infection by a pathogen.
  • LSU-P2 can be grown in brain heart infusion (BHI) containing 2% NaCI and kanamycin for selection if desired. Grows well on blood agar with kanamycin (50 micrograms/ml) for selection. Best growth at 23-27 degrees C, but requires 48 h to achieve a 2 mm colony.
  • BHI brain heart infusion

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Abstract

On décrit des vaccins vivants atténués dirigés l'un contre Edwardsiella ictaluri, l'autre contre Pasteurella piscicida. L'un et l'autre vaccins sont incapables de réversion relativement à la virulence, puisque tous deux sont obtenus par mutations par délétion dans le gène aroA, ou le gène purA, ou les deux. Lesdits vaccins peuvent être utilisés non seulement pour vacciner un poisson contre Edwardsiella ictaluri ou contre Pasteurella piscicida, mais également pour servir de vecteurs pour des antigènes présents issus d'autres ichtyopathogènes. Par conséquent, ces vaccins peuvent également être dirigés contre d'autres agents pathogènes sans risque d'infection par réversion relativement à la forme virulente de l'agent pathogène dans lequel l'antigène intervient naturellement.
PCT/US1998/007066 1997-04-11 1998-04-09 Vaccins vivants attenues diriges contre des ichtyopathogenes WO1998046725A2 (fr)

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EP98918062A EP0973865A2 (fr) 1997-04-11 1998-04-09 Vaccins vivants attenues diriges contre des ichtyopathogenes
IL13232798A IL132327A0 (en) 1997-04-11 1998-04-09 Attenuated invasive vaccines against fish pathogens
AU71055/98A AU7105598A (en) 1997-04-11 1998-04-09 Attenuated, invasive vaccines against fish pathogens
US09/402,695 US6350454B1 (en) 1997-04-11 1998-04-09 Attenuated Pasteurella piscicida vaccine for fish
IL132327A IL132327A (en) 1997-04-11 1999-10-11 Vaccines for fish containing weakened pastoralella piscicidae

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US9045742B2 (en) * 2009-05-29 2015-06-02 The Arizona Board Of Regents For And On Behalf Of Arizona State University Recombinant Edwardsiella bacterium
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